Potassium bromate as positive assay control for the Fpg-modified comet assay

Möller, Peter; Muruzabal, Damián; Bakuradze, Tamara; Richling, Elke; Bankoglu, Ezgi Eyluel; Stopper, Helga; Langie, Sabine A. S.; Azqueta, Amaya; Jensen, Annie Aarup; Scavone, Francesca; Giovannelli, Lisa; Wojewódzka, Maria; Kruszewski, Marcin; Valdiglesias, Vanessa; Laffón, Blanca; Costa, Carla; Costa, Solange; Teixeira, João Paulo; Marino, Mirko; Bò, Cristian Del; Riso, Patrizia; Shaposhnikov, Sergey; Collins, Andrew

doi:10.1093/mutage/geaa011

Cited by 33 publications

(23 citation statements)

References 28 publications

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“…hOGG1-modified comet assay has been widely used in human biomonitoring studies to detect oxidative DNA damage (Azqueta et al 2020). As it was previously described (Møller et al 2018), KBrO 3 causes oxidative damage to bases but do not generate DNA strand breaks, demonstrating that it is a suitable positive control for the enzyme-modified comet assay (Møller et al 2020b). Besides, KBrO 3 is known to induce hOGG1sensitive sites (Smith et al 2006;Pfuhler et al 2017).…”

Section: Discussionmentioning

confidence: 95%

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Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

et al. 2021

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Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

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Section: Discussionmentioning

confidence: 95%

“…Fresh and frozen saliva leucocytes and frozen PBL were exposed to four well-known DNA damaging agents: MMS, Act-D, ultraviolet (UV) radiation, and KBrO 3 . The doses used, as well as the treatment times, were selected on the basis of previous studies (Collins et al 1997;Sánchez-Flores et al 2015;Laffon et al 2017;Møller et al 2020b).…”

Section: Treatmentsmentioning

confidence: 99%

Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

et al. 2021

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“…ECVAG was superseded by the comet assay in human biomonitoring (hCOMET) project that has disseminated recommendations on technical procedures, , molecular epidemiological studies, and reporting of results . The hCOMET project also included a ring trial on the Fpg-modified comet assay, aiming at testing KBrO 3 as positive assay control …”

Section: Validity Of Methods For Detection Of Oxidatively Damaged Dnamentioning

confidence: 99%

“…Overall, the ESCODD ring trials show that interlaboratory variation (i.e., ratios between the highest and lowest values in background levels of 8-oxodG) increases as the sample becomes more complex, that is, 5.1-fold (8-oxodG standard in solution), 7.8-fold (calf thymus DNA), 10–100-fold (HeLa cells), 35-fold (PBMCs), and 100-fold (pig liver). ,− In comparison, the variation in Fpg-sensitive sites is <10-fold in cells that have been distributed from a central laboratory to partner laboratories. − The hCOMET trial assessed the variation in Fpg-sensitive sites in both reference samples (Ro19-8022 treated cells) and cells that were exposed to KBrO 3 in each of the participating laboratories and analyzed by a semistandardized comet assay procedure. The results showed a relatively high variation in the level of Fpg-sensitive sites in cells after KBrO 3 exposure (58-, 91-, 46-, and 13-fold between lowest and highest values after exposure to 0, 0.5, 1.5, and 4.5 mM, respectively) . However, all laboratories obtained a concentration–response relationship and found that the Ro19-8022 reference sample had levels of Fpg-sensitive sites that were within the range of the DNA damage of the KBrO 3 samples.…”

Section: Validity Of Methods For Detection Of Oxidatively Damaged Dnamentioning

confidence: 99%

Biomarkers of DNA Oxidation Products: Links to Exposure and Disease in Public Health Studies

Roursgaard

2021

Chem. Res. Toxicol.

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Environmental exposure can increase the production of reactive oxygen species and deplete cellular antioxidants in humans, resulting in oxidatively generated damage to DNA that is both a useful biomarker of oxidative stress and indicator of carcinogenic hazard. Methods of oxidatively damaged DNA analysis have been developed and used in public health research since the 1990s. Advanced techniques detect specific lesions, but they might not be applicable to complex matrixes (e.g., tissues), small sample volume, and large-scale studies. The most reliable methods are characterized by (1) detecting relevant DNA oxidation products (e.g., premutagenic lesions), ( 2) not harboring technical problems, (3) being applicable to complex biological mixtures, and (4) having the ability to process a large number of samples in a reasonable period of time. Most effort has been devoted to the measurements of 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxodG), which can be analyzed by chromatographic, enzymic, and antibody-based methods. Results from validation trials have shown that certain chromatographic and enzymic assays (namely the comet assay) are superior techniques. The enzyme-modified comet assay has been popular because it is technically simpler than chromatographic assays. It is widely used in public health studies on environmental exposures such as outdoor air pollution. Validated biomarker assays on oxidatively damaged DNA have been used to fill knowledge gaps between findings in prospective cohort studies and hazards from contemporary sources of air pollution exposures. Results from each of these research fields feed into public health research as approaches to conduct primary prevention of diseases caused by environmental or occupational agents.

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“…The photosensitizer Ro19-8022, which was used by the ESCODD and ECVAG ring trials, is not widely commercially available, and can be relatively difficult to find. Work performed after the ECVAG studies has identified potassium bromate as a good positive assay control for the hOGG1-and Fpg-modified comet assay [ 298 ]. The stability of potassium bromate-induced DNA damage in cryopreserved samples is currently being investigated, which would be a crucial aspect of any human biomonitoring or clinical study [ 298 ].…”

Section: The Futurementioning

confidence: 99%

Biomarkers of nucleic acid oxidation – A summary state-of-the-art

Chao

Evans

et al. 2021

Redox Biology

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Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2′-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2′-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo , but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease.

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Potassium bromate as positive assay control for the Fpg-modified comet assay

Cited by 33 publications

References 28 publications

Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

Biomarkers of DNA Oxidation Products: Links to Exposure and Disease in Public Health Studies

Biomarkers of nucleic acid oxidation – A summary state-of-the-art

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